Salmonella bacteriophage and antibacterial composition comprising the same

ABSTRACT

Disclosed herein is a novel bacteriophage which has specific bactericidal activity against one or more  Salmonella  bacteria selected from the group consisting of  Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum , and  Salmonella Pullorum  without affecting beneficial bacteria, in addition to showing excellent tolerance to acid, heat and desiccation. The novel bacteriophage can be widely used as an active ingredient for therapeutic agents, animal feeds or drinking water, cleaners and sanitizers for preventing and treating the infectious diseases caused by  Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum  or  Salmonella Pullorum  including salmonellosis,  Salmonella  food poisoning, Fowl Typhoid, and Pullorum disease or for controlling the  salmonella  bacteria.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/239,751 filed on Sep. 3, 2009, the disclosure of which is herebyexpressly incorporated by reference in its entirety and hereby expresslymade a portion of this application.

TECHNICAL FIELD

The present invention relates to a novel bacteriophage and antibacterialcomposition comprising the same.

BACKGROUND ART

Salmonella is a genus of the family Enterobacteriaceae, characterized asGram-negative, facultatively anaerobic, non spore-forming, rod-shapedbacteria, and most strains are motile by flagella. Salmonella has anaverage genomic GC content of 50-52%, which is similar to that ofEscherichia coli and Shigella. The genus Salmonella is a pathogenicmicroorganism that causes infections in livestock as well as in humans.Serological division has it that Salmonella enterica, a species ofSalmonella bacterium, has a variety of serovars including Gallinarum,Pullorum, Typhimurium, Enteritidis, Typhi, Choleraesuis, and derby (BoppC A, Brenner F W, Wells J G, Strokebine N A. Escherichia, Shigella,Salmonella. In Murry P R, Baron E J, et al., eds. Manual of ClinicalMicrobiology. 7th ed. Washington D.C. American Society for Microbiology1999; 467-74; Ryan K J. Ray C G (editors) (2004). Sherris MedicalMicrobiology (4th ed). McGraw Hill. ISBN 0-8385-8529-9.). Of them,Salmonella Gallinarum and Pullorum are fowl-adapted pathogens,Salmonella Typhi is a human-adapted pathogen, Salmonella Choleraesuisand Salmonella derby are swine-adapted pathogens, and SalmonellaEnteritis and Salmonella Typhimurium are pathogenic for humans andanimals. Each serovar causes illness in the respective species,resulting in tremendous damage to farmers or consumers.

A disease of domestic birds caused by Salmonella bacterium is FowlTyphoid (FT), which is caused by a pathogen, Salmonella Gallinarum(hereinafter, referred to as “SG”). Fowl Typhoid (FT) is a septicemicdisease of domestic birds such as chicken and turkey, and the course maybe acute or chronic with high mortality. A recent report has had it thatFowl Typhoid frequently occurs in Europe, South America, Africa, andSoutheast Asia, with damages increasing every year. Outbreaks of FT inSouth Korea have been reported since 1992 and economic losses caused byFT in brown, egg-laying chickens are very serious (Kwon Yong-Kook. 2000annual report on avian diseases. Information publication by NationalVeterinary Research & Quarantine Service. March, 2001; Kim Ae-Ran etal., The prevalence of pullorum disease-fowl typhoid in grandparentstock and parent stock in Korea, 2003, Korean J Vet Res (2006) 46(4):347˜353).

Pullorum disease is also caused by a strain of the Salmonella bacteria,Salmonella Pullorum (hereinafter, referred to as “SP”). Pullorum diseaseoccurs in any age or season, but young chickens are particularlysusceptible to the disease. During the past century, it has been aserious disease among young chickens at 1-2 weeks of age or younger.Since the 1980s, the occurrence has greatly decreased. However, it hasbeen growing since the mid-1990s (Kwon Yong-Kook. 2000 annual report onavian diseases. Information publication by National Veterinary Research& Quarantine Service. March, 2001; Kim Ae-Ran et al., The prevalence ofpullorum disease-fowl typhoid in grandparent stock and parent stock inKorea, 2003, Korean J Vet Res (2006) 46(4): 347˜353).

In South Korea, outbreaks of Fowl Typhoid and Pullorum disease have beenincreasing since the 1990s, inflicting economic damages on farmers. Forthis reason, a live attenuated SG vaccine has been used in broilers forthe prevention of Fowl Typhoid from 2004 (Kim Ae-Ran et al., Theprevalence of pullorum disease-fowl typhoid in grandparent stock andparent stock in Korea, 2003, Korean J Vet Res (2006) 46(4): 347˜353).Its efficacy is doubtful, and the live vaccine is not allowed to be usedfor layers because of the risk of egg-transmitted infections.Unfortunately, there are still no commercially available preventivestrategies against Pullorum disease, unlike Fowl Typhoid. Thus, there isan urgent need for new ways to prevent Fowl Typhoid and Pullorumdisease.

Meanwhile, Salmonella Enteritidis (hereinafter, referred to as “SE”) andSalmonella Typhimurium (hereinafter, referred to as “ST”) are zoonoticpathogens, which show no host specificity, unlike SG or SP (ZoobisesReport; United Kingdom 2003).

SE and ST are causative of salmonellosis in poultry, pigs, and cattle.Salmonellosis, caused by Salmonella bacteria, is an acute or chronicinfection of the digestive tract in livestock, and shows the majorsymptoms of fever, enteritis, and septicemia, occasionally pneumonia,arthritis, abortion, and mastitis. Salmonellosis occurs worldwide, andmost frequently during the summer months (T. R. Callaway et al.Gastrointestinal microbial ecology and the safety of the food supply asrelated to Salmonella. J Anim Sci 2008.86:E163-E172). In cattle, typicalsymptoms include loss of appetite, fever, dark brown diarrhea or bloodymucous in stool. The acute infection in calves leads to rapid death, andthe infection during pregnancy leads to fetal death due to septicemia,resulting in premature abortion. In pigs, salmonellosis is characterizedclinically by three major syndromes: acute septicemia, acute enteritis,and chronic enteritis. Acute septicemia occurs in 2˜4-month-old piglets,and death usually occurs within 2˜4 days after onset of symptoms. Acuteenteritis occurs during the fattening period, and is accompanied bydiarrhea, high fever, pneumonia, and nervous signs. Discoloration of theskin may occur in some severe cases. Chronic enteritis is accompanied bycontinuing diarrhea.

Once an outbreak of salmonellosis by SE and ST occurs in poultry, pigs,and cattle, it is difficult to cure only with therapeutic agents. Thereasons are that Salmonella bacteria exhibit a strong resistance tovarious drugs and live in cells that are impermeable to antibiotics uponthe occurrence of clinical symptoms. Up to now, there have been nomethods for effectively treating salmonellosis caused by SE and ST,including antibiotics.

As in livestock, SE and ST cause infections in humans via livestock andtheir products, leading to salmonella food poisoning. Intake ofinfected, improperly cooked livestock products (e.g., meat products,poultry products, eggs and by-products) infects humans. Salmonella foodpoisoning in humans usually involves the prompt onset of headache,fever, abdominal pain, diarrhea, nausea, and vomiting. The symptomscommonly appear within 6-72 hours after the ingestion of the organism,and may persist for as long as 4-7 days or even longer (NSW+HEALTH.2008.01.14.).

According to a report by the CDC (The Centers for Disease Control andPrevention, USA), 16% of human food poisoning outbreaks between 2005 and2008 were attributed to Salmonella bacteria, with SE and ST responsiblefor 20% and 18% thereof, respectively. With respect to salmonella foodpoisoning in humans between 1973 and 1984, the implicated food vehiclesof transmission were reportedly chicken (50), beef (19%), pork (7%),dairy products (6%), and turkey (9%). In 1974˜1984, the bacterialcontamination test on broilers during the slaughter process showed 35%or more of salmonella incidence. In 1983, salmonella was isolated in50.6% of chicken, 68.8% of turkey, 60% of goose, 11.6% of pork, and 1.5%of beef. Further, a survey carried out in 2007 reported that salmonellawas found in 5.5% of raw poultry meat and 1.1% of raw pork. Inparticular, it was revealed that SE commonly originated fromcontaminated egg or poultry meat, and ST from contaminated pork, poultrymeat, and beef (www.cdc.gov) (Centers for Disease Control and Prevention(CDC)). For example, food poisoning caused by SE has rapidly increasedin the US, Canada, and Europe since 1988, and epidemiological studiesdemonstrated that it was attributed to eggs or egg-containing foods(Agre-Food Safety Information Service (AGROS). Domestic and foreign foodpoisoning occurrence and management trend. 2008. 02). A risk assessmentconducted by FAO and WHO in 2002 noted that the human incidence ofsalmonellosis transmitted through eggs and poultry meat appeared to havea linear relationship to the observed Salmonella prevalence in poultry.This means that, when reducing the prevalence of Salmonella in poultry,the incidence of salmonellosis in humans will fall (Salmonella controlat the source; World Health Organization. International Food SafetyAuthorities Network (INFOSAN) Information Note No. 03/2007). Recently,fears about food safety have been spurred by outbreaks of salmonellafrom products as varied as peanuts, spinach, tomatoes, pistachios,peppers and, most recently, cookie dough (Jane Black and Ed O'Keefe.Overhaul of Food Safety Rules in the Works. Washington Post StaffWriters Wednesday, Jul. 8, 2009).

For these reasons, Salmonella infections must be reported in Germany (6and 7 of the German law on infectious disease prevention,Infektionsschutzgesetz). Between 1990 and 2005 the number of officiallyrecorded cases decreased from approximately 200,000 cases toapproximately 50,000. It is estimated that every fifth person in Germanyis a carrier of Salmonella. In the USA, there are approximately 40,000cases of Salmonella infection reported each year.

Therefore, there is an urgent need to control SE and ST, which causesalmonellosis in livestock and humans. The collaborative efforts of USDAand FDA have developed a number of effective strategies to preventsalmonellosis that causes over 1 million cases of food-borne illness inthe United States. Among them is a final rule, issued by the FDA, toreduce the contamination in eggs. The FDA will now require that eggproducers test regularly for lethal salmonella during egg production,storage and shipment. As a result, an estimated 79,000 illnesses and 30deaths due to contaminated eggs will be avoided each year (Jane Blackand Ed O'Keefe. Overhaul of Food Safety Rules in the Works. WashingtonPost Staff Writers Wednesday, Jul. 8, 2009). In Denmark, conservativeestimates from a cost benefit analysis comparing Salmonella controlcosts in the production sector with the overall public health costs ofsalmonellosis suggest that Salmonella control measures saved Danishsociety US$ 14.1 million in the year 2001 (Salmonella control at thesource. World Health Organization. International Food Safety AuthoritiesNetwork (INFOSAN) Information Note No. 03/2007).

Meanwhile, bacteriophage is a specialized type of virus that infects anddestroys only bacteria, and can self-replicate only inside hostbacteria. Bacteriophage consists of genetic material in the form ofsingle or double stranded DNA or RNA surrounded by a protein shell.Bacteriophages are classified based on their morphological structure andgenetic material. There are three basic structural forms ofbacteriophage according to morphological structure: an icosahedral(twenty-sided) head with a tail; an icosahedral head without a tail; anda filamentous form. Based on their tail structure, bacteriophages havingicosahedral head and double-stranded, linear DNA as their geneticmaterial are divided into three families: Myoviridae, Siphoviridae, andPodoviridae, which are characterized by contractile, longnoncontractile, and short noncontractile tails, respectively.Bacteriophages having an icosahedral head without a tail and RNA or DNAas their genetic material are divided based on their head shape andcomponents, and the presence of shell. Filamentous bacteriophages havingDNA as their genetic material are divided based on their size, shape,shell, and filament components (H. W. Ackermann. Frequency ofmorphological phage descriptions in the year 2000; Arch Virol (2001)146:843-857; Elizabeth Kutter et al. Bacteriophages biology andapplication; CRC press).

During infection, a bacteriophage attaches to a bacterium and insertsits genetic material into the cell. After this a bacteriophage followsone of two life cycles, lytic or lysogenic. Lytic bacteriophages takeover the machinery of the cell to make phage components. They thendestroy or lyse the cell, releasing new phage particles. Lysogenicbacteriophages incorporate their nucleic acid into the chromosome of thehost cell and replicate with it as a unit without destroying the cell.Under certain conditions, lysogenic phages can be induced to follow alytic cycle (Elizabeth Kutter et al. Bacteriophages biology andapplication. CRC Press).

After the discovery of bacteriophages, a great deal of faith wasinitially placed in their use for infectious-disease therapy. However,when broad spectrum antibiotics came into common use, bacteriophageswere seen as unnecessary due to a specific target spectrum.Nevertheless, the misuse and overuse of antibiotics resulted in risingconcerns about antibiotic resistance and harmful effects of residualantibiotics in foods (Cislo, M et al. Bacteriophage treatment ofsuppurative skin infections. Arch Immunol. Ther. Exp. 1987.2:175-183;Kim sung-hun et al., Bacteriophage; New Alternative Antibiotics.Biological research information center (BRIC)). In particular,antimicrobial growth promoter (AGP), added to animal feed to enhancegrowth, is known to induce antibiotic resistance, and therefore, the banof using antimicrobial growth promoter (AGP) has been recentlyintroduced. In the European Union, the use of all antimicrobial growthpromoters (AGPs) was banned from 2006. South Korea has banned the use ofsome AGPs from 2009, and is considering restrictions on the use of allAGPs in 2013˜2015.

These growing concerns about the use of antibiotics have led to aresurgence of interest in bacteriophage as an alternative toantibiotics. Seven bacteriophages for control of E. coli O157:H aredisclosed in U.S. Pat. No. 6,485,902 (Use of bacteriophages for controlof Escherichia coli O157, issued in 2002). Two bacteriophages forcontrol of various microorganisms are disclosed in U.S. Pat. No.6,942,858 (issued to Nymox in 2005). Many companies have been activelytrying to develop various products using bacteriophages. EBI food system(Europe) developed a food additive for preventing food poisoning causedby Listeria monocytogenes, named Listex-P100, which is the firstbacteriophage product approved by the US FDA. A phage-based product,LMP-102 was also developed as a food additive against Listeriamonocytogenes, approved as GRAS (Generally Regarded As Safe). In 2007, aphage-based wash produced by OmniLytics was developed to prevent E. coli0157 contamination of beef during slaughter, approved by USDA's FoodSafety and Inspection Service (FSIS). In Europe, Clostridium sporogenesphage NCIMB 30008 and Clostridium tyrobutiricum phage NCIMB 30008 wereregistered as a feed preservative against Clostridium contamination offeed in 2003 and 2005, respectively. Such studies show that researchinto bacteriophages for use as antibiotics against zoonotic pathogens inlivestock products is presently ongoing.

However, most of the phage biocontrol studies have focused on thecontrol of E. coli, Listeria, and Clostridium. Salmonella is also azoonotic pathogen, and damages due to this pathogen are not reduced. Asmentioned above, since SE and ST exhibit multiple drug resistance,nationwide antimicrobial resistance surveillance has been conducted inSouth Korea under the Enforcement Decree of the Act on the Prevention ofContagious Disease (Executive Order 16961), Enforcement ordinance of theAct on the Prevention of Contagious Disease (Ministry of Health andWelfare's Order 179), and Organization of the National Institute ofHealth (Executive Order 17164). Accordingly, there is a need for thedevelopment of bacteriophages to control Salmonella. The foregoingdiscussion is solely to provide background information of the inventionand do not constitute an admission of prior art.

SUMMARY

One aspect of the present invention relates to an isolatedbacteriophage, belonging to a morphotype group of the familySiphoviridae, with a specific bactericidal activity against one or moreSalmonella bacteria selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum, characterized by one of the following properties:the bacteriophage has a total genome size of 41˜43 kb; the bacteriophagecontains as a part of the genome thereof at least one nucleic acidsequence selected from among SEQ ID NOS. 1 to 6; and the bacteriophagehas major structural proteins ranging in size from 39 to 41 kDa and from16 to 18 kDa.

According to some embodiments, the foregoing bacteriophage may have amorphological structure composed of an isometric capsid and a long,non-contractile tail. According to some other embodiments, when PCR isperformed in a presence of a primer set selected from among SEQ ID NOS.7 and 8, SEQ ID NOS. 9 and 10, SEQ ID NOS. 11 and 12, SEQ ID NOS. 13 and14, SEQ ID NOS. 15 and 16, and SEQ ID NOS. 17 and 18, with the genome ofthe bacteriophage serving as a template, each PCR product may be 500bp˜1,000 bp long.

According to still some other embodiments, the foregoing bacteriophagemay show at least one of the following properties; tolerance to a rangeof from pH 3.0 to pH 11.0, tolerance to a heat range of from 37° C. to70° C. and tolerance to desiccation at 37˜60° C. for 0˜120 min. In oneembodiment, the bacteriophage may be identified by accession numberKCCM11027P.

Another aspect of the present invention relates to a composition forprevention or treatment of infectious diseases caused by one or moreSalmonella strains selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum, comprising the foregoing bacteriophage. In oneembodiment, the infectious disease may be salmonellosis and salmonellafood poisoning when caused by Salmonella enteritidis or SalmonellaTyphimurium, Fowl typhoid when caused by Salmonella Gallinarum andpullorum when caused by Salmonella Pullorum. In another embodiment, thecomposition may be used as an antibiotic.

Still another aspect of the present invention relates to an animal feedor drinking water, comprising the foregoing bacteriophage. Still anotheraspect of the present invention relates to a sanitizer and cleaner,comprising the foregoing bacteriophage.

Still another aspect of the present invention relates to a method forpreventing or treating infectious diseases caused by one or moreSalmonella strains selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum, comprising administering the foregoingbacteriophage to animals in need thereof. In some of certainembodiments, it is related to a method for preventing or treatinginfectious diseases caused by one or more Salmonella strains selectedfrom the group consisting of Salmonella Enteritidis, SalmonellaTyphimurium, Salmonella Gallinarum, and Salmonella Pullorum, comprisingadministering the foregoing composition to animals in need thereof.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Leading to the present invention, intensive and thorough research intobacteriophages, isolated from natural sources, which infect the poultrypathogen salmonella, conducted by the present inventors, aiming toovercome problems occurring upon the use of broad spectrum antibiotics,resulted in the finding that some isolated bacteriophages have aspecific bactericidal activity against Salmonella Enteritidis (SE),Salmonella Typhimurium (ST), Salmonella Gallinarum (SG) and SalmonellaPullorum (SP) with no influences on beneficial bacteria, in addition toshowing excellent acid- and heat-resistance and desiccation tolerance,as identified for the morphological, biochemical and genetic propertiesthereof, and thus that some bacteriophages are effective in theprevention and treatment of Salmonella Enteritidis- or SalmonellaTyphimurium-mediated diseases, such as livestock salmonellosis andSalmonella food poisoning, and Salmonella Gallinarum- or SalmonellaPullorum-mediated diseases, particularly, Fowl Typhoid and Pullorumdisease. Also, bacteriophages according to some embodiments of thepresent invention can be applied to various products for the control ofSalmonella bacteria, including livestock feed additives, drinking waterfor livestock, barn sanitizers, and cleaners for meal products. It isone aspect of the present invention to provide a novel bacteriophagewhich has a specific bactericidal activity against one or moreSalmonella bacteria selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum.

It is another aspect of the present invention to provide a compositionfor the prevention or treatment of infectious diseases caused by one ormore Salmonella bacteria selected from the group consisting ofSalmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,and Salmonella Pullorum, comprising the bacteriophage as an activeingredient.

It is a further aspect of the present invention to provide a livestockfeed additive, drinking water for livestock, and a cleaner or asanitizer, comprising the bacteriophage as an active ingredient.

It is still a further aspect of the present invention to provide amethod for preventing or treating infectious diseases caused by one ormore Salmonella bacteria selected from the group consisting ofSalmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,and Salmonella Pullorum using the composition comprising thebacteriophage as an active ingredient.

Having a specific bactericidal activity against one or more Salmonellabacteria selected from the group consisting of Salmonella Enteritidis,Salmonella Typhimurium, Salmonella Gallinarum, and Salmonella Pullorum,in addition to showing excellent acid- and heat-resistance anddesiccation tolerance, novel bacteriophages according to someembodiments of the present invention is effective for the prevention andtreatment of infectious diseases caused by Salmonella Enteritidis,Salmonella Typhimurium, Salmonella Gallinarum, or Salmonella Pullorum,including salmonellosis, Salmonella food poisoning, Fowl Typhoid andPullorum disease, and can also be used for the control of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum.

DESCRIPTION OF DRAWINGS

FIG. 1 is of photographs showing the formation of ΦCJ4 plaques in a lawnof salmonella bacteria: A: in a lawn of SE; B: in a lawn of ST; C: in alawn of SG; D: in a lawn of SP; E: in a lawn of SA; F: in a lawn of SB;G: in a lawn of SC; H: in a lawn of SD. Plaques formed in lawns of SE,ST, SG and SP, but not in lawns of SA, SB, SC and SD.

FIG. 2 is an electron microphotograph of ΦCJ4, showing that ΦCJ4 belongsto a morphotype group of the family Siphoviridae, characterized by anisometric capsid and a long non-contractile tail;

FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage ΦCJ4, inwhich major bands were detected at approximately 40 kDa and 17 kDa andminor bands at approximately 69 kDa, 38 kDa and 14 kDa, with See-blueplus 2 prestained-standard (Invitrogen) serving as a size marker;

FIG. 4 is the result of PFGE of the isolated bacteriophage ΦCJ4, showingthe total genome size of approximately 42 kb, with a 5 kbp DNA sizestandard (Bio-rad) serving as a size marker;

FIG. 5 is the result of PCR, performed using each primer set for theΦCJ4 genomic DNA: A: a 860 bp-long PCR product obtained with a primerset of SEQ ID NOS. 7 and 8; B: a 600 bp-long PCR product obtained with aprimer set of SEQ ID NOS. 9 and 10; C: a 900 bp-long PCR productobtained with a primer set of SEQ ID NOS. 11 and 12; D: a 950 bp-longPCR product obtained with a primer set of SEQ ID NOS. 13 and 14; E: a700 bp-long PCR product obtained with a primer set of SEQ ID NOS. 15 and16; F: a 880 bp-long PCR product obtained with a primer set of SEQ IDNOS. 17 and 18;

FIG. 6 is the result of acid-resistance assay on the bacteriophage ΦCJ4,showing the number of surviving bacteriophage at pH 2.5, 3.0, 3.5, 4.0,5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 10.0 and 11.0. The bacteriophage ΦCJ4 didnot lose its activity until pH 3.0, but completely lost its activity atpH 2.5 or lower, as compared to control;

FIG. 7 is the result of heat-resistance assay on the bacteriophage ΦCJ4,showing the number of surviving bacteriophage at 37, 45, 53, 60, 70 and80° C. for 120 min. The bacteriophage ΦCJ4 maintained its activity at upto 70° C.; and

FIG. 8 is the result of desiccation resistance assay on thebacteriophage ΦCJ4, performed at 60° C. for 120 min with the aid ofspeed vacuum dryer (SVD), in which when titer changes under the drycondition were measured in comparison with pre-drying titers, theactivity was decreased about 10-fold.

FIG. 9 is a graph in which body weights of rats are plotted against timeafter administration with single dosages of the bacteriophage ΦCJ4. ▪;male control administered with 20 mM Tris-HCl and 2 mM MgCl₂ mix, □;male control administered at a concentration of 1×10¹² pfu with ΦCJ4, ●;female control administered with 20 mM Tris-HCl and 2 mM MgCl₂ mix, ◯;female test group administered at a concentration of 1×10¹² pfu withΦCJ4. No significant changes in body weight were found even 14 daysafter the administration, in comparison with the control.

One embodiment of the present invention relates to a novelbacteriophage, belonging to a morphotype group of the familySiphoviridae, with a specific bactericidal activity against one or moreSalmonella bacteria selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium, Salmonella Gallinarum, andSalmonella Pullorum, having one of the following properties: 1) thebacteriophage has a total genome size of 41˜43 kb; 2) the bacteriophagecontains as a part of the genome thereof at least one nucleotidesequence selected from among SEQ ID NOS. 1 to 6; and 3) thebacteriophage has major structural proteins ranging in size from 39 to41 kDa and from 16 to 18 kDa.

In a preferred embodiment, the bacteriophage has a total genome size ofabout 42 kb, and structural proteins corresponding to respective sizesof about 69 kDa, about 40 kDa, about 38 kDa, about 17 kDa and about 14kDa. Further, the bacteriophage may contain as parts of the genomethereof nucleic acid molecules of SEQ ID NOS. 1 to 6.

Also, the bacteriophage according to some embodiments of the presentinvention has a morphological structure composed of an isometric capsidand a long, non-contractile tail.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA (gDNA and cDNA) and RNA molecules. The term “nucleotides” which whenjoined together, make up the structural units of nucleic acid molecules,encompass natural ones and sugar- or base-modified analogues thereof.

Further, the bacteriophage according to some embodiments of the presentinvention shows biochemical properties of being resistant to acid, heatand desiccation.

In greater detail, the bacteriophage according to some embodiments ofthe present invention has excellent resistance to acid and heat so thatit can survive over a wide pH range of from 3.5 to 11.0 and a heat rangeof from 37° C. to 70° C. With regard to the desiccation tolerancethereof, the bacteriophage can remain viable even under a hightemperature and dry condition (e.g., 120 min at 60° C.). Thanks to thesuperiority thereof in resistance to acid, heat and desiccation, thebacteriophage according to some embodiments of the present invention canbe used in a wide range of temperature and pH, finding applications incompositions and products for the prevention and treatment of livestockdiseases and livestock-mediated human diseases.

When PCR is performed in the presence of a primer set selected fromamong SEQ ID NOS. 7 and 8, SEQ ID NOS. 9 and 10, SEQ ID NOS. 11 and 12,SEQ ID NOS. 13 and 14, SEQ ID NOS. 15 and 16, and SEQ ID NOS. 17 and 18,with the genome of the bacteriophage according to some embodiments ofthe present invention serving as a template, each PCR product is 500bp˜1,000 bp long. Preferably, PCR product is 500 bp˜1,000 bp long, withthe primer sets of SEQ ID NOS. 7 and 8, SEQ ID NOS. 9 and 10, SEQ IDNOS. 11 and 12, SEQ ID NOS. 13 and 14, SEQ ID NOS. 15 and 16, and SEQ IDNOS. 17 and 18.

According to some embodiments, a bacteriophage was isolated from asewage sample of a chicken slaughterhouse that bacteriophage wasidentified as having a specific bactericidal activity against SE, ST, SGand SP and the above characteristics, and was designated as ΦCJ4 anddeposited with the Korean Culture Center of Microorganisms (361-221,Honje 1, Seodaemun, Seoul) on Aug. 14, 2009 under accession numberKCCM11027P.

In accordance with an example of the present invention, sewage sampleswere collected from chicken slaughterhouses and used to isolatetherefrom bacteriophages that can lyse the host cell SG. They were alsofound to lyse SE, ST, SG and SP (FIG. 1). An morphological examinationunder an electron microscope confirmed that the bacteriophage (ΦCJ4)belongs to a morphotype of the family Siphoviridae (FIG. 2).

The bacteriophage ΦCJ4 was found to have major structural proteins withthe size of 40 kDa and 17 kDa, as measured by a protein pattern analysis(FIG. 3).

Further, a genome analysis showed that ΦCJ4 has a total genome size ofapproximately 42 kbp (FIG. 4), with the nucleic acid molecules of SEQ IDNOS. 1 to 6 incorporated thereinto. Also, the bacteriophage was found tobe of very low genetic similarity with known bacteriophages as measuredby the comparison of genetic similarity with other species, indicatingthat the bacteriophage is a novel one. More particularly, when PCR wasperformed using the primer sets SEQ ID NOS. 7 and 8, SEQ ID NOS. 9 and10, SEQ ID NOS. 11 and 12, SEQ ID NOS. 13 and 14, SEQ ID NOS. 15 and 16,and SEQ ID NOS. 17 and 18, which were designed for ΦCJ4, the resultingPCR products were 860 bp, 600 bp, 900 bp, 950 bp, 700 bp, and 880 bp insize, respectively (FIG. 5).

Also, the phage plaques (clear zones formed in a lawn of cells on softagar due to lysis by phage) resulting from the infection of ΦCJ4 intoSE, ST, SG and SP were observed to have the same size and turbidity.

ΦCJ4 was examined for stability under a wide spectrum of pH,temperature, and desiccation. The bacteriophage was observed to surviveover a pH range of from 3.0 to 11.0 (FIG. 6) and a temperature range offrom 37° C. to 70° C. (FIG. 7) in addition to remaining stably viableeven after desiccation at high temperature (60° C. for 120 min) (FIG.8).

Also, the wild-type strains SE, ST, SG and SP were also found to fallwithin the host cell range of ΦCJ4.

When orally administered with ΦCJ4, rats were observed to remainunchanged in weight (FIG. 9), mortality, general symptoms and organabnormality.

Also, a claning assay shows that the bacteriophage ΦCJ4 is found to haveexcellent bactericidal activity against salmonella strains, compared toconventional cleaners as positive controls.

These data imply that the bacteriophage ΦCJ4 and its mutantscan beapplied to various products for the control of salmonella bacteria. Inaccordance with another aspect thereof, the present invention pertainsto a composition for the prevention or treatment of infectious diseasescaused by one or more Salmonella bacteria selected from the groupconsisting of Salmonella enteritidis, Salmonella Typhimurium, SalmonellaGallinarum, and Salmonella Pullorum, comprising the bacteriophage as anactive ingredient.

Preferably, examples of the infectious diseases include salmonellosisand Salmonella food poisoning by Salmonella enteritidis or SalmonellaTyphimurium, Fowl Typhoid by Salmonella Gallinarum and Pullorum diseaseby Salmonella Pullorum include, but are not limited thereto.

As used herein, the term “salmonellosis” refers to symptoms followingsalmonella infection, such as fever, headache, diarrhea, and vomiting.That is, salmonellosis is an infection with bacteria of the genusSalmonella, with the accompaniment of two representative symptoms:septicemia such as typhoid fever; and acute gastroenteritis such as foodpoisoning, enteritis, and acute bacteremia.

Having a specific bactericidal activity against Salmonella enteritidis,Salmonella Typhimurium, Salmonella Gallinarum and Salmonella Pullorumaccording to some embodiments, the bacteriophage can be used forpreventing or treating diseases that are caused by the bacteria. In apreferred embodiment, the composition may further comprise anantibiotic.

As used herein, the term “prevention” is intended to encompass allactions for restraining or delaying disease progress through theadministration of the composition. The term “treatment” in this contextencompasses all actions for improving or beneficially changing thepatient's condition through the administration of the composition.

According to some embodiments, the composition comprises ΦCJ4 in anamount of from 5×10² to 5×10¹² pfu/ml, and preferably in an amount offrom 1×10⁶ to 1×10¹° pfu/ml.

The composition according to some other embodiments of the presentinvention may further comprise a pharmaceutically acceptable vehicle,and may be formulated together with the carrier into foods, medicines,and feed additives.

As used herein, the term “pharmaceutically acceptable vehicle” refers toa carrier or diluent that neither causes significant irritation to anorganism nor degrades the biological activity and properties of theadministered active ingredient. For use in the formulation of thecomposition into a liquid preparation, a pharmaceutically acceptablevehicle must be suitable for sterilization and biocompatibility.Examples include saline, sterile water, Ringer's solution, bufferedphysiological saline, albumin infusion solution, dextrose solution,maltodextrin solution, glycerol, and ethanol. They may be used alone orin any combination thereof. If necessary, another conventional additive,such as antioxidants, buffers, bacteriostatic agents, etc., may be addedto the composition. When combined additionally with diluents,dispersants, surfactants, binders and/and lubricants, the compositionaccording to some embodiments of the present invention may be formulatedinto injections such as aqueous solutions, suspensions and emulsions, orpills, capsules, granules, or tablets.

The prophylactic or therapeutic compositions according to someembodiments of the present invention may be locally applied to afflictedareas by coating or spraying. Alternatively, the composition accordingto some other embodiments of the present invention may be administeredthrough oral or parenteral routes. The parenteral routes are availablefor intravenous, intraperitoneal, intramuscular, subcutaneous or topicaladministration

Depending on a variety of factors including formulations, the mode ofadministration, the age, weight, sex, condition and diet of the patientor animal being treated, the time of administration, the route ofadministration, the rate of excretion, and reaction sensitivity, thesuitable dosage of the composition according to some embodiments of thepresent invention will vary when it is applied, sprayed or administered.It will be apparent to those skilled in the art that when thepharmaceutical composition is administered to patients, the suitabletotal daily dose may be determined by an attending physician orveterinarian within the scope of sound medical judgment.

Oral dosage preparations of the composition according to someembodiments of the present invention may take the form of tablets,troches, lozenges, aqueous or emulsive suspensions, powders or granules,emulsions, hard or soft capsules, syrups, or elixirs. The oral dosageforms such as tablets and capsules may comprise a binder such aslactose, saccharose, sorbitol, mannitol, starch, amylopectin, celluloseor gelatin, an excipient such as dicalcium phosphate, a disintegrantsuch as corn starch or sweet potato starch, a lubricant such asmagnesium stearate, calcium stearate, sodium stearylfumarate, orpolyethylene glycol wax. For capsules, a liquid vehicle such as lipidmay be further used.

For non-oral administration, the composition according to someembodiments of the present invention may be formulated into injectionsvia subcutaneous, intravenous, or intramuscular routes, suppositories,or sprays inhalable via the respiratory tract, such as aerosols.Injection forms may be prepared by dissolving or suspending thecomposition according to some other embodiments of the presentinvention, together with a stabilizer or a buffer, in water and loadingthe solution or suspension onto ampules or vial unit forms. For sprays,such as aerosols, a propellant for spraying a water-dispersedconcentrate or wetting powder may be used in combination with anadditive.

The term “antibiotic”, as used herein, refer to a substance or compoundthat can be administered to animals to kill bacteria or inhibit theirgrowth and is intended to encompass antiseptics, bactericidal agents andantibacterial agents. The animals are mammals including humans. Thanksto the advantage of being of higher specificity for Salmonella overconventional antibiotics, the bacteriophage according to someembodiments of the present invention can kill the specific pathogenswithout affecting beneficial bacteria. Furthermore, the bacteriophageaccording to some other embodiments of the present invention does notinduce drug resistance so that it can be provided as a novel antibioticwith a long life cycle. In accordance with a further aspect thereof, thepresent invention pertains to an animal feed or drinking water,comprising the bacteriophage as an active ingredient.

Feed additive antibiotics used in the fishery and livestock industry areintended to prevent infections. However, most of the currently availablefeed additive antibiotics are problematic in that they are apt to inducethe occurrence of resistant strains and may be transferred to humans asthey remain in livestock products. The uptake of such residualantibiotics may make human pathogens resistant to antibiotics, resultingin the spread of diseases. Furthermore, many kinds of feed additiveantibiotics, usually used in combination in animal feeds, may cause theemergence of multidrug-resistant strains. Therefore, the bacteriophageaccording to some embodiments of the present invention can be used as afeed additive antibiotic that is eco-friendly enough to be a solution tothe problems.

The animal feed according to some embodiments of the present inventionmay be prepared by adding the bacteriophage directly or in a separatefeed additive form to an animal feed. In an animal feed, thebacteriophage according to some embodiments of the present invention maytake a liquid or a dry form, and preferably exist as a dried powder. Inthis regard, the bacteriophage according to some embodiments of thepresent invention may be dried by air drying, natural drying, spraydrying or freeze-drying, but these drying processes do not limit thepresent invention. The bacteriophage according to some embodiments ofthe present invention may be added as powder in an amount of from 0.05to 10% by weight, preferably in an amount of from 0.1 to 2% by weight,based on the total weight of animal feed. The animal feed may compriseother conventional additives useful for the preservation thereof for along term, in addition to the bacteriophage according to someembodiments of the present invention.

To the feed additive according to some embodiments of the presentinvention may be added another non-pathogenic microorganism. Theavailable additional microorganism may be selected from the groupconsisting of Bacillus subtilis that can produce protease, lipase andinvertase, Lactobacillus sp. strain that can exert physiologicalactivity and a function of decomposing under an aerobic conditions, suchas in the stomach of cattle, filamentous fungi including Aspergillusoryzae (J Animal Sci 43:910-926, 1976) that increases the weight ofdomestic animals, enhances milk production and helps the digestion andabsorptiveness of feeds, and yeast including Saccharomyces cerevisiae (JAnim Sci 56:735-739, 1983). The animal feed comprising ΦCJ4 inaccordance with some embodiments of the present invention may includeplant-based feeds, such as grains, nuts, food byproducts, seaweed,fiber, drug byproducts, oil, starches, meal, and grain byproducts, andanimal-based feeds such as proteins, minerals, fat, single cellproteins, zooplankton, and food wastes, but is not limited thereto.

The feed additive comprising ΦCJ4 in accordance with some otherembodiments of the present invention may include additives forpreventing quality deterioration, such as binders, emulsifiers andpreservatives, and additives for increasing utility, such as aminoacids, vitamins, enzymes, probiotics, flavorings, non-protein nitrogen,silicates, buffering agents, coloring agents, extracts, andoligosaccharides, but is not limited thereto.

When supplied with drinking water containing the bacteriophage accordingto some embodiments of the present invention, livestock can becontinuously reduced in the population of Salmonella bacteria in theintestine thereof livestock. As a result, Salmonella-free livestock canbe produced.

In accordance with still a further aspect thereof, the present inventionpertains to a cleaner or a sanitizer, comprising the bacteriophage as anactive ingredient.

The sanitizer comprising the bacteriophage as an active ingredient isvery useful for food hygiene against, for example, food poisoning. Indetail, the sanitizer may be utilized not only as an agent or a foodadditive for preventing salmonella contamination, but also in theproduction of salmonella-free livestock. In order to remove Salmonellar,the sanitizer can also be sprayed over domestic sewages and applied topoultry barns, slaughterhouses, spots where livestock died, cookingspaces and cooking facilities.

Further, the cleaner comprising the bacteriophage as an activeingredient can be used on a body area of living animals, such as skin,feathers and the like, which is already or potentially contaminated withSalmonella bacteria.

In accordance with still another aspect, the present invention relatesto a method for the prevention or treatment of infectious diseasescaused by one or more Salmonella bacteria selected from the groupconsisting of Salmonella Enteritidis, Salmonella Typhimurium, SalmonellaGallinarum, and Salmonella Pullorum using the bacteriophage or thecomposition.

The composition according to some embodiments of the present inventionmay be administered in the form of a pharmaceutical formulation intoanimals or may be ingested as a mixture with animal feed or drinkingwater by animals and preferably as a mixture with animal feed. In someembodiments of the present invention, the animals include cattle, pigs,chicken, poultry and humans, but are not limited thereto.

As long as it reaches target tissues, any route, whether oral orparenteral, may be taken for administering the composition according tosome embodiments of the present invention. In detail, the compositionaccording to some certain embodiments of the present invention may beadministered via oral, rectal, topical, intravenous, intraperitoneal,intramuscular, intraarterial, transdermal, intranasal, and inhalationroutes.

The method for the treatment of diseases in accordance with someembodiments of the present invention comprises administering thecomposition according to certain some embodiments of the presentinvention in a therapeutically effective amount. It is apparent to thoseskilled in the art that the total daily dose should be determined by anattending physician or veterinarian within the scope of sound medicaljudgment. The therapeutically effective amount for a given patient mayvary depending on various factors well known in the medical art,including the kind and degree of the response to be achieved, thepatient's age, body weight, state of health, sex, and diet, time androute of administration, the secretion rate of the composition, the timeperiod of therapy, concrete compositions according to whether otheragents are used therewith or not, etc.

A better understanding of some embodiments of the present invention maybe obtained through the following examples which are set forth toillustrate, but are not to be construed as limiting the presentinvention.

Example 1 Salmonella Bacteriophage Isolation

1-1. Bacteriophage Screening and Single Bacteriophage Isolation

50 ml of each sample from a chicken slaughterhouse, located in SuwonCity, Kyeonggi Do, South Korea, and a nearby sewage disposal plant weretransferred to a centrifuge tube, and centrifuged at 4000 rpm for 10min, followed by filtering the supernatant through a 0.45 μm filter. 18mL of the sample filtrate was mixed with 150 μl of a SalmonellaGallinarum (hereinafter referred to as “SG”) shaking culture medium(OD₆₀₀=2) and 2 ml of 10× Luria-Bertani medium (hereinafter referred toas “LB medium”) tryptone 10 g; yeast extract 5 g; NaCl 10 g; in a finalvolume of 1 L). The mixture was cultured at 37° C. for 18 hrs and thencentrifuged at 4000 rpm for 10 min after which the supernatant wasfiltered through a 0.2 μm filter. Separately, a mixture of 3 ml of 0.7%agar (w/v) and 150 μl of the SG shaking culture medium (OD₆₀₀=2) waspoured across an LB plate and allowed to solidify. Over this plate wasspread 10 μl of the culture filtrate, followed by incubation for 18 hrsat 37° C. (0.7% agar was used as “top-agar” and the titration of phagelysate was performed on the top-agar, called soft agar overlaytechnique).

A dilution of the sample culture medium containing the phage lysate wasmixed with 150 μL of an SG shaking culture medium (OD₆₀₀=2) andsubjected to soft agar overlay assay to produce single plaques. Since asingle plaque consisted of the same bacteriophage, one plaque was takenand dissolved in 400 μL of an SM solution (NaCl, 5.8 g; MgSO₄7H₂O, 2 g;1M Tris-Cl (pH7.5), 50 ml; H₂O, in a final volume of 1 L), and left for4 hours at room temperature to isolate single bacteriophages. To amplifythe isolated bacteriophage, 100 μL of the supernatant was taken from thesingle bacteriophage solution, mixed with 12 mL of 0.7% agar and 500 μLof an SG shaking culture medium, and subjected to a soft agar overlayassay on an LB plate (150 mm in diameter). 15 mL of an SM solution waspoured to a plate in which lysis had been completed, after which theplate was gently shaken for 4 hrs at room temperature to elute thebacteriophages from the top-agar. The SM solution containing the elutedbacteriophages was recovered, and chloroform was added in an amountcorresponding to 1% of the final volume, and mixed well for 10 min.After centrifugation at 4000 rpm for 10 minutes, the resultingsupernatant was filtered through a 0.2 μm filter, and stored in therefrigerator until use.

1-2. Large-Scale Batches of Bacteriophage

The selected bacteriophage was cultured at a large scale using SG. SGwas cultured with shaking. After an aliquot of 1.5×10¹⁰ cfu (colonyforming units) was centrifuged at 4000 rpm for 10 min, the pellet wasre-suspended in 4 ml of an SM solution. Into the suspension wasinoculated 7.5×10⁷ pfu (plaque forming unit) of the bacteriophage at anMOI (multiplicity of infection) of 0.005), followed by incubation at 37°C. for 20 min. This solution was inoculated into 150 mL of an LB mediain a flask, and cultured at 37° C. for 5 hrs. Chloroform was added in anamount corresponding to 1% of the final volume before the culturesolution was shaken for 20 min. DNase I and RNase A were added to afinal concentration of 1 μg/ml, each. The solution was left at 37° C.for 30 min. NaCl and PEG (polyethylene glycol) were added to a finalconcentration of 1 M and 10% (w/v), respectively and left at 4° C. foran additional 3 hrs. The solution was centrifuged at 4° C. and 12,000rpm for 20 min to discard the supernatant. A suspension of the pellet in5 mL of an SM solution was left at room temperature for 20 minutes andmixed well with 4 mL of chloroform. After centrifugation at 4° C. and4000 rpm for 20 min, the supernatant was filtered through a 0.2 μmfilter and then subjected to ultracentrifugation using a glyceroldensity gradient to purify ΦCJ4 (density: 40%, 5% glycerol at 35,000 rpmand 4° C. for 1 hr). The purified ΦCJ4 was re-suspended in 300 μL of anSM solution, followed by titration. ΦCJ4 was deposited with the KoreanCulture Center of Microorganisms (361-221, Honje 1, Seodaemun, Seoul) onAug. 14, 2009 under accession number KCCM11027P.

Example 2 Examination on ΦCJ4 Infection of Salmonella

To analyze the selected bacteriophage for lytic activity on Salmonellaspecies other than SG, attempts were made of cross infection with otherSalmonella species. As a result, ΦCJ4 did not infect SC (Salmonellaenterica Serotype Choleraesuis), SD (Salmonella enterica SerotypeDerby), SA (Salmonella enterica sub sp. Arizonae), and SB (Salmonellaenterica sub sp. Bongori), but infected SE (Salmonella Enteritidis), ST(Salmonella Typhimurium), SG (Salmonella Gallinarum) and SP (SalmonellaPullorum) (see Example 12). The results are summarized in Table 1, belowand shown in FIG. 1.

TABLE 1 ΦCJ4 Infection of Salmonella Sero Strain Plaque Sero StrainPlaque type name formation type name formation SE SGSC 2282 ◯ SA ATCC13314 X ST ATCC 14028 ◯ SB ATCC 43975 X SG SGSC 2293 ◯ SC ATCC 10708 XSP SGSC 2295 ◯ SD ATCC 6960 X ATCC: The Global BioresourceCenter SGSC:Salmonella Genetic Stock Center

Example 3 Morphology of Bacteriophage ΦCJ4

The purified ΦCJ4 was diluted in a 0.01% gelatin solution, and thenfixed in a 2.5% glutaraldehyde solution. The sample was dropped onto acarbon-coated mica plate (ca. 2.5×2.5 mm), adapted for 10 min, andwashed with sterile distilled water. A carbon film was mounted on acopper grid, stained with 4% uranyl acetate for 30-60 sec, and dried.Examination under a JEM-1011 transmission electron microscope (at 80 kV,magnification of ×120,000˜×200,000) had it that the purified ΦCJ4consisted morphologically of an isometric capsid and a longnon-contractile tail, indicating that it belongs to a morphotype groupof the family Siphoviridae.

Example 4 Protein Pattern Analysis of ΦCJ4

15 μL of a ΦCJ3 solution purified at a titer of 10¹¹ pfu/ml was mixedwith 3 μL of a 5×SDS sample solution, and heated for 5 min. The totalprotein of ΦCJ4 was run on 4˜12% NuPAGE Bis-Tris gel (Invitrogen). Then,the gel was stained with Coomassie blue for 1 hr at room temperature.Major bands were detected at 40 kDa and 17 kDa with the appearance ofother bands at 69 kDa, 38 kDa and 14 kDa, as shown in FIG. 3.

Example 5 Total Genomic DNA Size of ΦCJ4

Genomic DNA of ΦCJ4 was isolated using ultracentrifugation. In thisregard, to a purified ΦCJ4 culture medium were added EDTA(ethylenediaminetetraacetic acid (pH8.0)), proteinase K, and SDS (sodiumdodecyl sulfate) at a final concentration of 20 mM, 50 ug/ml, and 0.5%(w/v), respectively, followed by incubation at 50° C. for 1 hr. An equalvolume of phenol (pH 8.0) was added and mixed well. After centrifugationat 12,000 rpm and room temperature for 10 min, the supernatant was mixedwell with an equal volume of PCI(phenol:chloroform:isoamylalhocol=25:24:1). Another centrifugation at12,000 rpm and room temperature for 10 min produced a supernatant whichwas then mixed with 1/10 volume of 3 M sodium acetate and two volumes ofcold 95% ethanol, and left at −20° C. for 1 hr. After centrifugation at0° C. and 12,000 rpm for 10 min, the supernatant was completely removed,and the DNA pellet was dissolved in 50 μL of TE (Tris-EDTA (pH 8.0)).The extracted DNA was diluted 10-fold, and measured for absorbance atOD₂₆₀ to determine its concentration 1 μg of the total genomic DNA wasloaded onto 1% PFGE (pulse-field gel electrophoresis) agarose gel andelectrophoresed at room temperature for 20 hrs with the aid of a BIO RADPFGE system program 7 (size range 25-100 kbp; switch time ramp 0.4-2.0seconds, linear shape; forward voltage 180 V; reverse voltage 120V). Asshown in FIG. 4, the genomic DNA of ΦCJ4 was approximately 42 kb long.

Example 6 Genetic Analysis of ΦCJ4

The genetic analysis of the purified ΦCJ4 started with triple digesting1 μg of the genomic DNA of ΦCJ4 with the restriction enzymes, EcoR V,Sca I and Nru I. Separately, another triple digestion was performed withthe restriction enzymes StuI, PvuII, and HincII. The vector pCL1920(Promega) was digested with Sma I, and treated with CIP (calf intestinalalkaline phosphatase). The digested genomic DNA was mixed at a ratio of3:1 with the vector, and ligated at 16° C. for 5 hrs. The resultingrecombinant vector was transformed into E. coli DH5α which was thenplated on an LB plate containing kanamycin and X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for blue/whiteselection. The selected colonies were cultured for 16 hrs in a culturemedium containing kanamycin with shaking. Then, plasmids were extractedusing a plasmid purification kit (Promega).

The cloning of the plasmids was confirmed by PCR using a primer set ofFRT135 and FRT136 (SEQ ID NOS. 19 and 20, respectively), and selectionwas made only of insert fragments having a size of 1 kb or longer. Theirbase sequences were analyzed using the primer set of FRT135 and FRT136(SEQ ID NOS. 19 and 20, respectively). The base sequences thus obtainedwere given in SEQ ID NOS. 1 to 6, respectively, and analyzed forsequence similarity with the aid of a NCBI blastx program, and theresults are summarized in Table 2, below.

TABLE 2 Sequence Similarity between ΦCJ4 and Other BacteriophagesAccession Subject Query E No organism protein number location locationIdentity value 1 Salmonella hypothetical YP_308656 187-528   3-1022286/347 6e−124 phage KS7 protein BPKS7gp56 (82%) 2 Salmonellahypothetical YP_224063.1 |  1-212  80-715 202/212 1e−94 phage KS7protein BPKS7gp41 (95%) Salmonella DNA polymerase YP_001110810  1-212 80-715 198/212 4e−91 phage SETP3 (93%) 3 Salmonella hypotheticalYP_308656 187-479  3-881 274/293 3e−139 phage KS7 protein BPKS7gp56(93%) 4 Salmonella hypothetical YP_224072  1-180 540-1  172/180 5e−95phage KS7 protein BPKS7gp50 (95%) Salmonella tailspike proteinYP_001110804  1-180 540-1  171/180 3e−94 phage SETP3 (95%) Salmonellatail component YP_001110803 467-598 951-556 124/132 1e−66 phage SETP3protein (93%) Salmonella hypothetical YP_224073 578-709 951-556 122/1323e−65 phage KS7 protein BPKS7gp51 (92%) 5 Salmonella hypotheticalYP_308657  1-96 567-854 95/96 8e−56 phage KS7 protein BPKS7gp57 (98%)Salmonella hypothetical YP_308658  1-78 196-429 77/78 1e−13 phage KS7protein BPKS7gp58 (98%) 6 Salmonella tail component YP_001110803 201-531 1-993 324/331 6e−124 phage SETP3 protein (97%) Salmonella hypotheticalYP_224073 312-642  1-993 322/331 1e−123 phage KS7 protein BPKS7gp51(97%)

Example 7 Design of ΦCJ4-Specific Primer Sequences

In order to identify ΦCJ4, ΦCJ4-specific primers were designed on thebasis of SEQ ID NOS. 1 to 6. PCR was performed using each primer set ofSEQ ID NOS. 7 and 8, SEQ ID NOS. 9 and 10, SEQ ID NOS. 11 and 12, SEQ IDNOS. 13 and 14, SEQ ID NOS. 15 and 16 and SEQ ID NOS. 17 and 18. 0.1 μgof the genomic DNA of bacteriophage and 0.5 pmol of each primer wereadded to pre-mix (Bioneer), and the final volume was adjusted to 20 μL.PCR was performed with 30 cycles of denaturation; 94° C. 30 sec,annealing; 60° C. 30 sec, and polymerization; 72° C., 1 min. The PCRproducts thus obtained were approximately 860 bp, 600 bp, 900 bp, 950bp, 700 bp, and 880 bp long, respectively, with the primer sets of SEQID NOS. 7 and 8, SEQ ID NOS. 9 and 10, SEQ ID NOS. 11 and 12, SEQ IDNOS. 13 and 14, SEQ ID NOS. 15 and 16 and SEQ ID NOS. 17 and 18. Theresults are shown in FIG. 5.

Example 8 pH Stability of Bacteriophage

In order to determine whether ΦCJ4 survives the low pH environment inthe stomach of livestock, ΦCJ4 was assayed for stability in a wide rangeof pH (pH 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.2, 9.0, 10.0, 11.0).Various pH solutions (sodium acetate buffer (pH 4.0, pH 5.5, pH 6.4),sodium citrate buffer (pH 2.5, pH 3.0, pH 3.5), sodium phosphate buffer(pH 6.9, pH 7.4) and Tris-HCl (pH 8.2, pH 9.0, pH 10.0, pH 11.0)) wereprepared to have a concentration of 2 M. 180 μL of each pH solution wasmixed with 20 μL of a bacteriophage solution (1.0×10¹⁰ pfu/ml), followedby incubation at room temperature for 2 hr. The reaction solution wasserially diluted, and 10 μL of each dilution was cultured at 37° C. for18 hrs by a soft agar overlay method to determine the titers of thephage lysates. Titer changes with pH were measured to determine thestability of bacteriophage over pH in comparison to titers of ΦCJ4 at 0hr. The results showed that the bacteriophage did not lose its activityand remained stable down to pH 3.5. The results are shown in FIG. 6.

Example 9 Heat Stability of Bacteriophage

For use as a feed additive, the bacteriophage was assayed for stabilityto the heat generated during a formulation process. In this regard, 200μL, of a ΦCJ4 solution with a titer of 1.0×10¹⁰ pfu/ml was incubated at37° C., 45° C., 53° C., 60° C., 70° C., or 80° C. for 2 hr. The solutionwas serially diluted, and 10 μL, of each dilution was cultured at 37° C.for 18 hrs by a soft agar overlay method to determine the titers ofphage lysates. Titer changes with temperature and exposure time weremeasured to determine the stability of bacteriophage to heat incomparison to titers at 0 hr and 37° C. The results showed that thebacteriophage did not lose its activity at 70° C. for up to 2 hrs, butwas deactivated at 80° C. or higher. The results are shown in FIG. 7.

Example 10 Desiccation Tolerance of Bacteriophage

For use as a feed additive, the bacteriophage was assayed for toleranceto the dry condition set for a formulation process. On the basis of theresults obtained from the heat stability assay, an assay was performedat 60° C. for 2 hr as follows. 200 μL of a ΦCJ4 solution (1.0×10¹¹pfu/ml) was dried using a Speed vacuum (Speed-Vacuum Concentrator 5301,Eppendorf). The pellet thus obtained was completely re-suspendedovernight at 4° C. in an equal volume of an SM solution. The solutionwas serially diluted, and 10 μL of each dilution was cultured at 37° C.for 18 hrs using a soft agar overlay method to determine the titers ofphage lysates. Titer changes under the dry condition were measured todetermine the desiccation tolerance of the bacteriophage in comparisonwith pre-drying titers. The results showed that its activity wasdecreased about 10-fold. The results are shown in FIG. 8.

Example 11 Spectrum of Wile-Type Host Cell Strains to whichBacteriophage Infects

ΦCJ4 was assayed for lytic activity against Korean wild-type SE (38strains), ST (22 strains), SG (56 strains) and SP (19 strains), obtainedfrom Laboratory of Avian Diseases, College of Veterinary Medicine, SeoulNational University, and National Veterinary Research and QuarantineService and the Korea Centers for Disease Control and Prevention, inaddition to the strains used in at least some embodiments of the presentinvention, SE (SE SCSG 2282), ST (ST ATCC14028), SG (SG SGSC2293) and SP(SP SGSC2295). 150 μL of each strain shaking culture medium (OD₆₀₀=2)was mixed, and 10 μL of ΦCJ4 solution (10¹⁰ pfu/ml) was cultured at 37°C. for 18 hrs using a soft agar overlay method to monitor the formationof plaques. It was observed that the bacteriophage ΦCJ4 showed lyticactivity of 97% against SE, 91% against ST, 96% against SG and 85%against SP. The results are summarized in Table 3, below.

TABLE 3 Lytic Activity of ΦCJ4 against Korean Wild-Type Strains SE, ST,SG, and SP Plaque Plaque Sero- Strain for- Sero- Strain for- type namemation type name mation SG SNU SG1 ◯ ST SNU ST1 ◯ SNU SG2 ◯ SNU ST2 ◯SNU SG3 ◯ SNU ST3 ◯ SNU SG4 ◯ SNU ST4 ◯ SNU SG5 ◯ SNU ST7 ◯ SNU SG6 ◯SNU ST8 ◯ SNU SG7 ◯ SNU ST11 ◯ SNU SG8 ◯ SNU ST12 ◯ SNU SG9 ◯ SNU ST13 ◯SNU SG10 ◯ SNU ST14 ◯ SNU SG11 ◯ SNU ST17 ◯ SNU SG12 ◯ SNU ST18 X SNUSG13 ◯ SNU ST19 X SNU SG14 ◯ SNU ST20 ◯ SNU SG15 ◯ SNU ST25 ◯ SNU SG16 ◯SNU ST26 ◯ SNU SG17 ◯ SNU ST37 ◯ SNU SG18 ◯ SNU ST38 ◯ SNU SG19 ◯ SNUST41 ◯ SNU SG20 ◯ SNU ST42 ◯ SNU SG21 ◯ ATCC UK1 ◯ SNU SG22 ◯ ATCC14028S ◯ SNU SG23 ◯ SGSC STM1412 ◯ SNU SG24 ◯ SGSC STM260 ◯ SNU SG25 ◯SGSC STM SA2197 ◯ SNU SG26 ◯ SE SGSC SE2282 ◯ SNU SG27 ◯ SGSC SE2377 ◯SNU SG28 ◯ PT4 S1400194 ◯ SNU SG30 ◯ PT4 LA52 ◯ SNU SG31 ◯ NVRQS SE004 ◯SNU SG32 ◯ NVRQS SE005 ◯ SNU SG33 ◯ KCDC SE008 ◯ SNU SG34 ◯ KCDC SE009 ◯SNU SG36 ◯ KCDC SE010 ◯ SNU SG37 ◯ KCDC SE011 X SNU SG38 ◯ KCDC SE012 ◯SNU SG39 ◯ KCDC SE013 ◯ SNU SG40 ◯ KCDC SE014 ◯ SNU SG41 ◯ KCDC SE015 ◯SNU SG42 ◯ KCDC SE016 ◯ SNU SG43 ◯ KCDC SE017 ◯ SNU SG44 ◯ KCDC SE018 ◯SNU SG45 ◯ KCDC SE019 ◯ SNU SG46 ◯ KCDC SE020 ◯ SNU SG47 ◯ KCDC SE021 ◯SNU SG48 ◯ KCDC SE022 ◯ SNU SG49 ◯ KCDC SE023 ◯ SNU SG50 ◯ KCDC SE024 ◯SGSC SG9184 ◯ KCDC SE025 ◯ SGSC SG2292 ◯ KCDC SE026 ◯ SGSC SG2293 ◯ KCDCSE027 ◯ SGSC SG2744 ◯ KCDC SE028 ◯ SGSC SG2796 ◯ KCDC SE029 ◯ SP SNU SP1◯ KCDC SE030 ◯ SNU SP4 ◯ KCDC SE031 ◯ SNU SP5 ◯ KCDC SE032 ◯ SNU SP8 ◯KCDC SE033 ◯ SNU SP11 ◯ KCDC SE034 ◯ SGSC SP2294 ◯ KCDC SE035 ◯ SGSCSP2295 ◯ KCDC SE036 ◯ SGSC SP2737 X KCDC SE037 ◯ SGSC SP2739 X SC ATCCSC10708 X SGSC SP2742 ◯ ATCC SC2929 X SGSC SP2743 ◯ SD ATCC SD6960 XSGSC SP2745 ◯ ATCC SD2466 ◯ SGSC SP2751 ◯ ATCC SD2467 ◯ SGSC SP4663 XATCC SD2468 X SGSC SP4664 ◯ SA ATCC SA13314 X SGSC SP4665 ◯ SB ATCCSB43975 X SGSC SP4666 ◯ SGSC SP4667 ◯ SGSC SA1684 ◯ SNU: Laboratory ofAvian Diseases, College of Veterinary Medicine, Seoul NationalUniversity SGSC: salmonella genetic stock center NVRQS: NationalVeterinary Research & Quarantine Service KCDC: Korean Centers forDisease Control and prevention ATCC: The Global BioresourceCenter

Example 12 Toxicity Assay of Bacteriophage

For safety use in the prevention of salmonellosis, salmonella foodpoisoning, fowl typhoid and pullorum, the bacteriophage was in vivoassayed for toxicity. Toxicity assay was performed with single oraldosages. In this assay, rats were orally administered with a singledosage of ΦCJ4 and monitored for acute toxicity to determine approximatelethal concentrations of ΦCJ4. To this end, first, specific-pathogenfree (SPF) male and female rats (SD) 7 weeks old, each of 10, werestarved for 24 hrs before administration with ΦCJ4. On theadministration day, five males and five females were orally administeredat a dose of 10 mL/kg with ΦCJ4 having a titer of 1×10¹² pfu/ml using anoral sonde while five controls were orally administered with a 20 mMTris-HCl and 2 mM MgCl₂ mix. Four hrs after the oral administration,feeds were provided for rats. Monitoring was conducted every hour for 4hours, starting from 30 min after the administration on the day ofadministration. Since then, they were monitored once a day for 14 daysfor general symptoms. None of them died. Neither toxic symptoms nornoticeable clinical symptoms were generated by ΦCJ4. The results aresummarized in Tables 4 and 5, below. Body weights were recorded beforeand 1 3, 7, 10 and 14 days after administration. No significant changeswere observed in body weight, indicating that ΦCJ4 does not cause atoxic reaction sufficient to reduce appetite or to change the bodyweight. These results are shown in FIG. 9. No noticeable abnormalitieswere found in any organ as examined by autopsy and with the naked eye.Therefore, the novel bacteriophage ΦCJ4 is non-toxic.

TABLE 4 Oral Toxicity Assay of ΦCJ4 in Terms of Mortality Hours afterDays after Done treatment treatment Final Sex (Pfu/kg) 0.5 1 2 3 4 5 6 78 9 10 11 12 13 14 Mortality Male Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00/5 10¹³ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 Female control 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0/5 10¹³ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5

TABLE 5 Oral Toxicity Assay of ΦCJ4 in Terms of General Symptoms DoneFinal Mortality Clinical Signs Sex Pfu/kg Male Female Male Female MaleControl 0/5 0/5 0/5 0/5 10¹³ 0/5 0/5 0/5 0/5 Female Control 0/5 0/5 0/50/5 10¹³ 0/5 0/5 0/5 0/5

TABLE 6 Oral Toxicity Assay of ΦCJ4 in Terms of Organ Abnormality DoneSex (pfu/kg) Gross finding Frequency Male Control N.A.D 5/5 10¹³ N.A.D5/5 Female Control N.A.D 5/5 10¹³ N.A.D 5/5 a: not detected.

Example 13 Efficiency of Bacteriophage as a Cleaner

In order to evaluate the efficiency of the bacteriophage as a cleanerfor meat products, the bacteriophage was assayed for ability to controlsalmonella bacteria in comparison with a conventional cleaner. In thisregard, 50 g of chicken breast cuts was purchased from a store. An STshaking culture (O.D.=2) was adjusted to a concentration of 10⁸ cfu/mland uniformly spread in an amount of 200 μL over the chicken breast cutswhich were then dried at room temperature for 12 min. The bacteriophageΦCJ4 and the cleaner chlorine (4˜6% Sodium hypochlorite) were containedat a concentration of 10⁸ pfu/L and 50 ppm in respective sprayers andsprayed at a rate of one stroke/sec for 10 sec. The treated chickenbreast cuts were placed in respective sanitary packs to which an SMbuffer was then added. The packs were shaken in a semicircle pattern.The WCR (whole carcass rinse) thus obtained was serially diluted and thedilutions were spread over LB media, followed by incubation at 37° C.for 18 hrs to determine the number of ST. When treated with thebacteriophage ΦCJ4, the population of ST was 24.29% reduced. Incontrast, the cleaner was observed to induce salmonella bacteria toincrease 14%. Accordingly, the bacteriophage ΦCJ4 has cleaning activityfar better than that of the conventional cleaner. The results are givenin Table 7, below.

TABLE 7 Comparison of Cleaning Efficiency between ΦCJ4 and CleanerSubstance Reduction Rate of Salmonella (%) 1 SM buffer 2 Chlorine 50 ppm(+14.50) 3 ΦCJ4 2.0 × 10⁸ pfu/L 24.49

Having specific bactericidal activity against one or more Salmonellabacteria selected from the group consisting of Salmonella Enteritidis(SE), Salmonella Typhimurium (ST), Salmonella Gallinarum (SG), andSalmonella Pullorum (SP) without affecting beneficial bacteria, inaddition to showing excellent tolerance to acid, heat and desiccation,as described hitherto, the novel bacteriophage according to someembodiments of the present invention can be widely used as an activeingredient for therapeutic agents, animal feeds or drinking water,cleaners and sanitizers for preventing and treating the infectiousdiseases caused by Salmonella Enteritidis, Salmonella Typhimurium,Salmonella Gallinarum or Salmonella Pullorum including salmonellosis,Salmonella food poisoning, Fowl Typhoid, and Pullorum disease.

Although preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1. An isolated bacteriophage having a specific bactericidal activityagainst one or more Salmonella bacteria selected from the groupconsisting of Salmonella Enteritidis, Salmonella Typhimurium, SalmonellaGallinarum, and Salmonella Pullorum, which is deposited under accessionnumber KCCM11027P.
 2. A composition for prevention or treatment ofinfectious diseases caused by one or more Salmonella bacteria selectedfrom the group consisting of Salmonella Enteritidis, SalmonellaTyphimurium, Salmonella Gallinarum, and Salmonella Pullorum, comprisingthe bacteriophage of claim
 1. 3. The composition according to claim 2,wherein the infectious diseases are salmonellosis and salmonella foodpoisoning caused by Salmonella enteritidis or Salmonella Typhimurium,Fowl typhoid caused by Salmonella Gallinarum and pullorum caused bySalmonella Pullorum.
 4. An antibiotic composition comprising an isolatedbacteriophage having a specific bactericidal activity against one ormore Salmonella bacteria selected from the group consisting ofSalmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,and Salmonella Pullorum, which is deposited under accession numberKCCM11027P.
 5. An animal feed or drinking water, comprising thebacteriophage of claim
 1. 6. A sanitizer and cleaner, comprising thebacteriophage of claim
 1. 7. A method for treating infectious diseasescaused by one or more Salmonella bacteria selected from the groupconsisting of Salmonella Enteritidis, Salmonella Typhimurium, SalmonellaGallinarum, and Salmonella Pullorum, comprising administering thebacteriophage of claim 1 to animals in need thereof.
 8. A method fortreating infectious diseases caused by one or more Salmonella bacteriaselected from the group consisting of Salmonella Enteritidis, SalmonellaTyphimurium, Salmonella Gallinarum, and Salmonella Pullorum, comprisingadministering the composition of claim 2 to animals in need thereof.